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Investigators probing the collapse of MF Global Holdings Ltd. are scrutinizing two money transfers made during the securities firm's final days in an effort to uncover what happened to $1.6 billion in missing customer funds.

Federal regulators at the Commodity Futures Trading Commission and the U.S. bankruptcy trustee for MF Global's brokerage unit are examining two separate transfers from customer accounts, including a previously undisclosed $165 million transaction, said people familiar with the matter. They said some of the investigators are poring over emails and records related to these and other money transfers.

MF Global collapsed into bankruptcy on Oct. 31, hurt by repercussions from an outsize bet its chief executive, Jon S. Corzine, made on bonds of troubled European countries.

Among those at the center of money transfers in the days leading up to the collapse was Edith O'Brien, an assistant treasurer at MF Global.

On the morning of Oct. 28, according to people familiar with the matter, Ms. O'Brien received a call from Mr. Corzine, who asked her to resolve a problem: The firm's bank account at J.P. Morgan Chase & Co. in London had run out of money, creating an overdraft.

After the call, Ms. O'Brien sent an email to about a dozen colleagues. "I need $175 mm sent immediately," said the message, a copy of which was reviewed by The Wall Street Journal.

Money to cover the overdraft was transferred to the London bank account later that day from MF Global's customer account, according to regulators and former MF Global officials.

As the chaos mounted at MF Global, Ms. O'Brien stopped leaving the office at night, napping on a bench in between long spells of trying to juggle transfer demands.

Mr. Corzine later thrust Ms. O'Brien into the spotlight when he told a congressional panel in December that she had assured him the $175 million transfer was proper.

Federal rules for futures trading allow brokerage firms to keep some of their own money in customer accounts, for reasons that include providing clients with ease of trading, and this money can be withdrawn. But customer funds are never to be touched.

Ms. O'Brien later declined to sign documents presented by J.P. Morgan affirming that the $175 million transfer had been proper, said people with knowledge of the matter.

Prosecutors and lawmakers have sought to meet with Ms. O'Brien, but she has so far declined, asking for legal immunity before agreeing to talk with them, people familiar with the matter said.

Neither Ms. O'Brien, Mr. Corzine nor other MF Global officials have been accused of any wrongdoing.

This article is based on a review of internal MF Global emails and documents, as well as interviews with people with knowledge of the circumstances surrounding the securities firm's frantic final days.

The emails and other documents reviewed by the Journal disclose new details about the $175 million transfer as well as revealing the other transaction—for $165 million—that may have contributed to the shortfall in accounts of MF Global customers.

J.P. Morgan, as a clearing bank for MF Global, played a part in both transfers. So did officials in MF Global's Chicago office, where Ms. O'Brien and several dozen others worked in relative obscurity while Mr. Corzine—a former Goldman Sachs Group Inc. chairman and later a senator and governor in

New Jersey—sought to remake the sleepy commodities firm into a risk-hungry trading house. Ms. O'Brien was among those responsible for moving funds when customer accounts were tapped, said people familiar with details of the securities firm's demise.

To keep track of both customer and firm money, MF Global relied partly on a computer spreadsheet that had to be updated manually.

The rush of activity in MF Global's final days caused many more erroneous trades than the firm would normally have in a similar period, according to James Giddens, the bankruptcy trustee in charge of MF Global's U.S. brokerage unit, who has raised concerns about whether anything close to the $1.6 billion total now estimated to be missing can be recovered.

Ms. O'Brien and some of her MF Global colleagues didn't realize there was a shortfall in customer accounts until it was too late, according to people familiar with the matter. They added that the Chicago employees expected that some trades happening in those final days would bring cash back to the firm's customer accounts. But not all the funds returned as anticipated.

On Wednesday, Oct. 26, five days before MF Global's collapse, officials in Chicago approved a $165 million transfer between two units of the firm around 5 p.m. Central time, the internal documents show.

Shortly after, $165 million arrived at an MF Global account at J.P. Morgan, and these funds had been pulled from the customer account, according to the documents and people familiar with the transfer.

Oct. 26 was the first day MF Global customer accounts showed a shortfall, according to the bankruptcy trustee.

Two days later, MF Global officials tried to reverse the transfer from the customer account, documents show. The result of those efforts is unclear.

By that day—Friday, Oct. 28—the situation had grown worse. Ms. O'Brien was staying at work all night, napping occasionally on a bench in her office, a person familiar with the matter said.

That Friday morning, when Mr. Corzine called about the overdraft in the London J.P. Morgan account, he told Ms. O'Brien that unless the overdraft was remedied, J.P. Morgan wouldn't make any more trades for MF Global, depriving the securities firm of incoming cash.

When $175 million subsequently landed in the J.P. Morgan account, it may have not been clear whether the funds belonged to customers, since the customer account handled both MF Global money and customer money, according to people familiar with the situation.

After J.P. Morgan received the money to remedy the overdraft, Mr. Corzine called Ms. O'Brien again, telling her the bank wanted assurances the transfer hadn't improperly come from customer money, people familiar with the situation said. Ms. O'Brien received a letter originally sent from the bank asking for a signature acknowledging that the funds had been moved properly.

MF Global's general counsel, Laurie Ferber, and Dennis Klejna, assistant general counsel, worked with Ms. O'Brien to try to find a version she was comfortable signing, but were unsuccessful, people familiar with the situation said. Ms. O'Brien and others felt the letter was too broadly written, these people said.

The next day, J.P. Morgan sent a revised letter addressed to Ms. O'Brien, a copy of which was reviewed by the Journal. She never signed the letter, according to people familiar with the matter.

Mr. Corzine and others received daily reports about the customer accounts, specifically information about how much of the firm's available money sat alongside clients' funds. Mr. Corzine has said he didn't know of any deficit in customer funds until hours before the Monday, Oct. 31, bankruptcy filing.